Growth of organ primordia during post-gastrula morphogenesis, along with cell shape change and cell differentiation, leads to the form and shape of the developing tissue. Although cell replication occurs throughout the organ forming period in most tissues, its role in generating the force for forming the three dimensional shape of the tissue such as the evagination of a tube from an epithelial sheet, is not well understood. Factors that have hindered comprehension of the potential of cell division for shape generation include the previous inability to experimentally separate mitotic pressure from other shape generating processes and the lack of a system in which the process can be followed in situ. In the proposed study the role of cell replication in generating both the number and pattern of cells in the epithelium of a segment, as a stimulant for cell differentiation of specific cell types within the epithelium, and as a motive force in the shape change of the epithelium during limb bud formation will be investigated using larvae of the brine shrimp, Artemia. The simplicity of the tissue organization of these larvae makes this organism ideally suited for this study. The cells involved in the limb bud and segment forming regions are few in number, clearly defined, not surrounded by other cells, and have well timed growth periods. Analysis of the experiments will be carried out using both video-based image analysis and immunocytochemistry. The following hypotheses will be tested: 1) Cell replication occurs in a spatio-temporally directed manner that results in a set pattern of cells and regions of unequal cell density. 2) Attainment of this pattern leads to the differentiation of arthrodial membrane and tendonal cells in which microtubules are involved in cell shape change. 3) Deformation of the epithelium and evagination of the thoracopod limb bud result form unequal cell density, the presence of differentiated arthrodial membrane cells, and region-specific apolysis. 4) Maintenance of the form and shape of the limb bud involves continued patterned cell replication, new cuticle formation, and tendonal cell differentiation. The role of microtubules and microfilaments in the evagination process will also be determined. Some sections of the study will provide preliminary evidence necessary for future studies on regulation of cell replication during development of segmental structures.